Construction and efficacy of a new live chimeric C-strain vaccine with DIVA characteristics against classical swine fever

To develop the new classical swine fever (CSF) vaccine candidate with differentiating infected vaccinated animals (DIVA) characteristics, a chimeric CSF virus (CSFV) was constructed based on an infectious cDNA clone of the CSF vaccine C-strain. The 5’- and 3’-untranslated regions (UTRs) and partial E2 region (residues 690-860) of the C-strain were substituted with the corresponding regions of bovine viral diarrhoea virus (BVDV) to construct the chimeric cDNA clone pC/bUTRs-tE2. The chimeric virus rC/bUTRs-tE2 was generated by several passages of pC/bUTRs-tE2-transfected PK15 cells. Stable growth and genetic properties of rC/bUTRs-tE2 were obtained after 30 serial passages. Compared to parental rC/bUTRs-tE2 (1^st passage), two residue mutations (M834K and M979K) located in E2 in rC/bUTRs-tE2 P30 were observed. Compared to the C-strain, rC/bUTRs-tE2 exhibited unchanged cell tropism and decreased plaque-forming ability. Substituting the C-strain UTRs with the BVDV UTRs resulted in significantly increased viral replication in PK15 cells. Compared to CSFV E^rns-positive and BVDV tE2-negative antibody responses induced by the CSF vaccine C-strain, immunization of rabbits and piglets with rC/bUTRs-tE2 resulted in serological profiles of CSFV E^rns- and BVDV tE2-positive antibodies, which are used to serologically discriminate pigs that are clinically infected and vaccinated. Vaccination of piglets with rC/bUTRs-tE2 conferred complete protection against lethal CSFV challenge. Our results suggest that rC/bUTRs-tE2 is a promising new CSF marker vaccine candidate.     

The feasibility of a novel bioreactor for vaccine production of classical swine fever virus

The performance of a new type of tide mode culture system was investigated in this study. This novel bioreactor provides two separated stages, liquid and gas, for cell growth requirements. The immobilized cells absorbed the nutrient from medium during the liquid stage and subsequently were exposed directly to fresh air to absorb oxygen during the gas stage. Operating with PK15 cells under optimal conditions, we obtained 2.3 × 10⁹ cells in 500 ml reactor. It is 30 times higher than the initial inoculum and about 11 times higher than the production by roller bottle. For the vaccine production of classical swine fever (CSFV), a high virus titer of 2.1 × 10⁹ median tissue culture infective dose (TCID₅₀) was yielded which provided exceed 300 doses per milliliter of CSFV solution. Therefore, this new cultural system performed well not only for cell production but also for virus yield. It should be a highly efficient production for the CSFV vaccine and have practical potential in other animal cell culture vaccine.     

Safety of classical swine fever virus vaccine strain LOM in pregnant sows and their offspring

The present study aimed to evaluate the safety of the classical swine fever virus (CSFV) vaccine strain LOM in pregnant sows. Pregnant sows with free CSFV antibody were inoculated with a commercial LOM vaccine during early pregnancy (day 38; n = 3) or mid-pregnancy (days 49–59; n = 11). In pregnant sows vaccinated during the early stages of gestation, abortion (day 109) was observed in one case, with two stillbirths and seven mummified fetuses. The viability of live-born piglets was 34.9% in sows vaccinated during mid-pregnancy compared with 81.8% in the control group. Post-mortem examination of the organs of the sows and piglets did not reveal any pathological lesions caused by CSFV; however, CSFV RNA was detected in the organs of several vaccinated sows and their litters. The LOM strain was transmitted from sows with free CSFV antibody to their fetus, but did not appear to induce immune tolerance in the offspring from vaccinated pregnant sows. Side effects were not observed in pregnant sows with antibody to the LOM strain: transmission from sow to their litters and stillbirth or mummified fetuses. The LOM strain may induce sterile immunity and provide rapid, long-lasting, and complete protection against CSFV; however, it should be contraindicated in pregnant sows due to potential adverse effects in pregnant sows with free CSFV antibody.     

Innocuousness and safety of classical swine fever marker vaccine candidate CP7_E2alf in non-target and target species

Chimeric pestivirus CP7_E2alf is a promising live marker vaccine candidate against classical swine fever. Prior to a possible application in the field, several safety aspects have to be addressed. Due to the fact that CP7_E2alf is based on a bovine viral diarrhea virus backbone, its behavior in ruminants is of particular interest. In the framework of this study, its innocuousness in non-target species was addressed by inoculation of calves, young goats, lambs, and rabbits. To this means, high titres of CP7_E2alf were applied orally to three animals of each species. Additional animals were left as unvaccinated contact controls. During the study, all animals remained clinically healthy, and neither fever nor leukopenia were observed. Virus could not be isolated from purified white blood cells or from nasal or faecal excretions. Moreover, none of the animals (inoculated or contact control) seroconverted.In the target species, innocuousness, shedding and transmission of vaccine virus was addressed in different animal trials that were carried out primarily for the purpose of efficacy, potency or duration of immunity studies. In all experiments, CP7_E2alf proved to be completely safe for the vaccinees and unvaccinated contact controls. Furthermore, no shedding or transmission was detected in any of the experiments. Even after parental vaccination, vaccine virus genome was barely detectable in blood or organ samples of vaccinated animals. Thus, CP7_E2alf can be regarded as completely safe for both target and non-target species.     

The impact of porcine circovirus associated diseases on live attenuated classical swine fever vaccine in field farm applications

Porcine circovirus associated diseases (PCVADs) are among the most important diseases affecting the worldwide swine industry. Vaccination against porcine circovirus type 2 (PCV2) infection has been utilized for disease control and effectively reduces clinical signs of PCVADs. To evaluate the efficacy of the PCV2 vaccine in field farms, we conducted a trial using conventional pigs immunized with the subunit PCV2 vaccine followed by PCV2 challenge. Immunized pigs demonstrated lower serum viral loads, less viral antigen staining in lymph nodes, and higher average daily weight gain, confirming the protective efficacy of the vaccine. However, low levels of PCV2 infection were still detected in vaccinated pigs after challenge, suggesting that the PCV2 vaccine was unable to eradicate the virus, which could lead to asymptomatic PCV2 subclinical infection (PCV2-SI) in pig farms. Additionally, PCV2 infection is a risk factor for impaired pig immune response development during the weaning to growth stages, which is a crucial period to receive vaccines against classical swine fever (CSF). Therefore, the impact of PCV2-SI or PCV2-systemic disease (PCV2-SD) on live attenuated CSF vaccine was investigated. After PCV2 challenge, there was no difference in levels of classical swine fever virus (CSFV) neutralizing antibodies (NA) between pigs with PCV2-SD and PCV2-SI, suggesting that the efficacy of CSF vaccine was compromised. Moreover, results of long-term monitoring of CSFV NA titers in PCV2-SI pigs with minimized interference by maternally-derived antibodies suggested that serum PCV2 viral loads greater than 10² copies/mL may compromise the efficacy of CSF vaccine. Overall, a conventional pig model was established to demonstrate the impaired efficacy of the subunit PCV2 vaccine and its impact on the CSF vaccine in vaccination-challenge trials. Additionally, the impaired efficacy of the PCV2 vaccine resulted in increased PCV2-SI, eventually leading to compromised the live attenuated CSF vaccine induced NA response in field farm applications.     

Modified live marker vaccine candidate CP7_E2alf provides early onset of protection against lethal challenge infection with classical swine fever virus after both intramuscular and oral immunization

Due to the vast economic consequences of classical swine fever (CSF) outbreaks, emergency vaccination plans are under discussion in European Union Member States. However, animals vaccinated with the conventional C-strain vaccine are subject to trade restrictions. To ease these restrictions, potent marker vaccines are required. One promising candidate is the chimeric pestivirus CP7_E2alf. For emergency vaccination in a CSF outbreak scenario, early onset of immunity is required. Here, the studies performed with a CP7_E2alf virus stock produced under good manufacturing conditions (GMP) are reported. In challenge experiments, CP7_E2alf induced full clinical protection 1 week after intramuscular vaccination, and 2 weeks after oral immunization. Furthermore, even after application of diluted vaccine preparations complete protection could be achieved if challenge infection was carried out 4 weeks after vaccination. In conclusion, GMP-produced CP7_E2alf proved to be a suitable marker vaccine candidate – also for emergency vaccination – both after intramuscular and oral application.     

A new subgenotype 2.1d isolates of classical swine fever virus in China, 2014

The lapinized attenuated vaccine against classical swine fever (CSF) has been used in China for over half a century and has generally prevented large-scale outbreaks in recent years. However, since late 2014, a large number of new cases of CSF were detected in many immunized pig farms in China. Several of these CSV viruses were isolated and characterized. Phylogenetic and genomic sequence analyses indicate that these new isolates, as well as some reference isolates, form a new subgenotype named 2.1d, and share several consistent molecular characteristics. Since these new isolates emerged in disparate geographic regions within 5 months, this suggests that these isolates may be widespread. Given that current vaccines do not appear to provide effective protection against this new subgenotype, further investigation of these strains is urgently needed.     

Partial protection against classical swine fever virus elicited by dendrimeric vaccine-candidate peptides in domestic pigs

We report the immunogenicity of three dendrimeric peptide vaccine candidates for classical swine fever virus (CSFV). Each dendrimeric construct contained four copies of a B-cell epitope from the E2 glycoprotein of CSFV [construct 1: E2 (694-712); 2: E2 (712-727); 3: E2 (829-842)] joined to a T-cell epitope from the NS3 protein (residues 1446-1460). Intramuscular immunization of domestic pigs with the different constructs significantly reduced the clinical score after lethal challenge with CSFV. In contrast, control pigs developed severe clinical signs of the disease. All pigs vaccinated with construct 1, containing a B-cell epitope from the E2 B-C domain, developed an antibody response that recognized not only the original dendrimeric immunogen but also its constituting E2 epitope in linear form, albeit no neutralizing antibodies were detected prior to viral challenge. Two of these pigs were partially protected, which associated with the induction of IFN-γ producing cells and of neutralizing antibodies upon challenge. Interestingly, the serological response elicited by construct 1 lacked antibodies to E2 A domain, used as infection markers. The dendrimeric approach could therefore provide a basis for the development of CSFV marker (DIVA) vaccines, and contribute to a better understanding of the immune responses against CSFV.     

First assessment of classical swine fever marker vaccine candidate CP7_E2alf for oral immunization of wild boar under field conditions

Oral vaccination against classical swine fever (CSF) is a potent tool to control disease outbreaks in wild boar. So far, vaccination campaigns have been carried out using live attenuated vaccines that do not allow serological differentiation of infected from vaccinated animals (DIVA). Although this drawback is acceptable for wild boar, the use of marker vaccines would facilitate studies on disease and vaccination dynamics. Recently, the CSF marker vaccine candidate CP7_E2alf was assessed for oral immunization under laboratory conditions. Promising results prompted efforts to study the vaccine candidate under field conditions and in bait formulation. In this context, two oral vaccination campaigns were carried out with CP7_E2alf bait vaccines in two areas called ‘faunistic-hunting farms’ in the region of Umbria, Italy. One campaign was conducted using single vaccination, the second with the routinely employed double vaccination strategy. Both campaigns were carried out before concerted hunting actions were performed. Bait uptake, vaccine virus detection and antibody responses were assessed along with inspections upon gutting. As a comparator, seven wild boar were hand-fed with baits under laboratory conditions. In the field, bait uptake ranged from 63.7% to 98.7%, whereas antibody prevalence reached only 33.3–35.1%. The marker serology showed a strong influence of sample quality on the test outcome with a total of 85% of samples being classified correctly. Vaccine virus was not detectable. Under hand feeding conditions, six out of seven wild boar took up at least one bait, and five of them showed detectable antibody levels seven weeks after vaccination. These results were supplemented by stability tests. Appropriate stability of vaccine virus was shown both under field and laboratory conditions. In total, most results were in line with our expectations. However, optimization of the DIVA assay has to be attempted in the future.     

A novel alphavirus replicon-vectored vaccine delivered by adenovirus induces sterile immunity against classical swine fever

Low efficacy of gene-based vaccines due to inefficient gene delivery and expression has been major bottleneck of their applications. Efforts have been made to improve the efficacy, such as gene gun and electroporation, but the strategies are difficult to put into practical use. In this study, we developed and evaluated an adenovirus-delivered, alphavirus replicon-vectored vaccine (chimeric vector-based vaccine) expressing the E2 gene of classical swine fever virus (CSFV) (rAdV-SFV-E2). Rabbits immunized with rAdV-SFV-E2 developed CSFV-specific antibodies as early as 9 days and as long as 189 days and completely protected from challenge with C-strain. Pigs immunized with rAdV-SFV-E2 (n = 5) developed robust humoral and cell-mediated responses to CSFV and were completely protected from subsequent lethal CSFV infection clinically and virologically. The level of immunity and protection induced by rAdV-SFV-E2 was comparable to that provided by the currently used live attenuated vaccine, C-strain. In contrast, both the conventional alphavirus replicon-vectored vaccine pSFV1CS-E2 and conventional adenovirus-vectored vaccine rAdV-E2 provided incomplete protection. The chimeric vector-based vaccine represents the first gene-based vaccine that is able to confer sterile immunity and complete protection against CSFV. The new-concept vaccination strategy may also be valuable in vaccine development against other pathogens.     

A single dose of the novel chimeric subunit vaccine E2-CD154 confers early full protection against classical swine fever virus

Classical swine fever is an economically important, highly contagious disease of swine worldwide. Subunit vaccines are a suitable alternative for the control of classical swine fever. However, such vaccines have as the main drawback the relatively long period of time required to induce a protective response, which hampers their use under outbreak conditions. In this work, a lentivirus-based gene delivery system is used to obtain a stable recombinant HEK 293 cell line for the expression of E2-CSFV antigen fused to porcine CD154 as immunostimulant molecule. The E2-CD154 chimeric protein was secreted into the medium by HEK293 cells in a concentration around 50 mg/L in suspension culture conditions using spinner bottles. The E2-CD154 immunized animals were able to overcome the challenge with a high virulent CSF virus strain performed 7 days after a unique dose of the vaccine without clinical manifestations of the disease. Specific anti-CSFV neutralizing antibodies and IFN-γ were induced 8 days after challenge equivalent to 14 days post-vaccination. The present work constitutes the first report of a subunit vaccine able to confer complete protection by the end of the first week after a single vaccination. These results suggest that the E2-CD154 antigen could be potentially used under outbreak conditions to stop CSFV spread and for eradication programs in CSF enzootic areas.     

Yeast expressed classical swine fever E2 subunit vaccine candidate provides complete protection against lethal challenge infection and prevents horizontal virus transmission

Classical swine fever (CSF) caused by the classical swine fever virus (CSFV) is a highly contagious swine disease resulting in large economical losses worldwide. The viral envelope glycoprotein E(rns) and E2 are major targets for eliciting antibodies against CSFV in infected animals. A Pichia pastoris yeast expressed E2 protein (yE2) has been shown to induce a protective immune response against CSFV challenge. The purpose of this study is to determine the optimal dose of yE2 and its efficacy on the prevention of virus horizontal transmission. A yeast-expressed E(rns) (yE(rns)) protein was also included to evaluate its immunogenicity. The yE(rns) vaccinated pigs seroconverted to CSFV-E(rns)-specific antibody but no neutralizing antibody was detected and none survived after challenge infection, suggesting yE(rns) and yE2 retain correct immunogenicity but only the yE2 is able to induce a protective immune response. All three doses of yE2 (200, 300, and 400μg) could elicit high titers of neutralizing antibodies and protective responses after challenge. The yE2/200 group demonstrated a mild fever response but recovered soon, and none of the yE2/300 and yE2/400 pigs became febrile. The optimal dose of yE2 was recommended to be 300μg of the total amount of secreted proteins. In addition, the yE2 vaccine could cross-protect from all three genotypes of viruses. Further, the yE2 vaccine efficacy in preventing virus horizontal transmission was evaluated by cohabitation of unimmunized sentinels 3 days after challenge infection. All the sentinel pigs were alive and had no clinical symptoms confirming yE2 vaccine could confer a protective immune response and prevent horizontal transmission of CSFV.     

Comprehensive evaluation of the adenovirus/alphavirus-replicon chimeric vector-based vaccine rAdV-SFV-E2 against classical swine fever

Classical swine fever (CSF) is an economically important, highly contagious swine disease caused by classical swine fever virus (CSFV). Marker vaccines and companion serological diagnostic tests are thought to be a promising strategy for future control and eradication of CSF. Previously, we have demonstrated that an adenovirus-vectored Semliki forest virus replicon construct expressing the E2 glycoprotein from CSFV, rAdV-SFV-E2, induced sterile immunity against a lethal CSFV challenge. In this study, we further evaluated the vaccine with respect to its safety, number and dose of immunization, and effects of maternally derived antibodies, re-immunization of the vaccine or co-administration with pseudorabies vaccine on the vaccine efficacy. The results showed that: (1) the vaccine was safe for mice, rabbits and pigs; (2) two immunizations with a dose as low as 6.25 × 10⁵ TCID₅₀ or a single immunization with a dose of 10⁷ TCID₅₀ rAdV-SFV-E2 provided complete protection against a lethal CSFV challenge; (3) maternally derived antibodies had no inhibitory effects on the efficacy of the vaccine; (4) the vaccine did not induce interfering anti-vector immunity; and (5) co-administration of rAdV-SFV-E2 with a live pseudorabies vaccine induced antibodies and protection indistinguishable from immunization with either vaccine administered alone. Taken together, the chimeric vaccine represents a promising marker vaccine candidate for control and eradication of CSF.     

Vaccination influences the evolution of classical swine fever virus

Classical swine fever is a serious, economically damaging disease caused by classical swine fever virus (CSFV). The CSFV is composed of two clades, according to phylogenetic estimates. Attenuated live vaccine such as HCLV, has been widely used to protect pigs from CSFV, but the influence of vaccination on the evolution of CSFV has not been studied. We conducted a systemic analysis of the impact of vaccination on the evolution of CSFV by comparing vaccine-related and non-vaccine-related CSFV groups. We found that vaccination may affect strain diversity and immune escape through recombination and point mutation. We also found that vaccination may influence the population dynamics, evolutionary rate and adaptive evolution of classical swine fever virus. Our evidence suggests that the vaccination might also change host adaptation through influencing codon usage of the virus in swine. These findings suggest that it is necessary to avoid excessive use of CSFV attenuated vaccines.     

Assessment of the efficacy of an attenuated live marker classical swine fever vaccine (Flc-LOM-BErns) in pregnant sows

Here, we constructed an attenuated live marker classical swine fever (CSF) vaccine (Flc-LOM-BE^rns) to eradicate CSF. This was done by taking infectious clone Flc-LOM, which is based on an attenuated live CSF vaccine virus (LOM strain), and removing the full-length classical swine fever virus (CSFV) E^rns sequences and the 3′ end (52 base pairs) of the CSFV capsid. These regions were substituted with the full-length bovine viral diarrhoea virus (BVDV) E^rns gene sequence and the 3′ end (52 base pairs) of the BVDV capsid gene. Sows were vaccinated with the Flc-LOM-BE^rns vaccine 3 weeks before insemination and then challenged with virulent CSFV at the early, mid- or late stages of pregnancy. We then examined transplacental transmission to the foetuses. Piglets born to sows vaccinated with Flc-LOM-BE^rns did not show vertical infection, regardless of challenge time. In addition, CSFV challenge did not affect the delivery date, weight or length of the foetus. Pregnant sows inoculated with the Flc-LOM-BE^rns vaccine were anti-CSF E^rns antibody-negative and anti-BVDV E^rns antibody-positive. Challenge of pregnant sows with virulent CSFV resulted in anti-CSF E^rns antibody positivity. These results strongly indicate that differential diagnosis can be conducted between the Flc-LOM-BE^rns vaccinated animal and virulent CSFV affected animal by detecting antibody against BVDV E^rns or CSF E^rns gene. Therefore, the Flc-LOM-BE^rns vaccine may fulfil the function of differential diagnosis which required for DIVA vaccine.     

Phylogenetic analysis using E2 gene of classical swine fever virus reveals a new subgenotype in China

Outbreaks of classical swine fever (CSF) have caused serious economic consequences in China. Phylogenetic analysis based on full-length E2 gene sequences showed that five classical swine fever virus (CSFV) isolates collected from Hunan province in 2011 and 2012, together with seven other isolates from neighboring provinces, Guangdong (5) and Guangxi (2), could be classified as a new subgenotype 2.1c, which may have been endemic in the south of China for at least fourteen years. Subgenotype 2.1c isolates share 90.2–94.9% and 89.9–93.8% nucleotide sequence similarity separately with those of subgenotype 2.1a and 2.1b in E2 gene, which are lower than the nucleotide identities between subgenotype 2.1a and 2.1b (91.1–95.7%). Further analysis based on a partial E2 gene sequence (216 nt) indicated that subgenotype 2.1c isolates are also circulating in Thailand. Alignment of E2 amino acid sequences showed that subgenotype 2.1c isolates exhibit a SPA → TPV substitution at positions 777 and 779 compared with subgenotypes 2.1a and 2.1b.     

Efficacy of marker vaccine candidate CP7_E2alf in piglets with maternally derived C-strain antibodies

Marker vaccines offer the possibility to differentiate classical swine fever (CSF) infected from CSF vaccinated animals based on serology and their implementation will ensure free trade with pigs. Therefore, new generations of promising marker vaccines have been developed, among them the chimeric vaccine CP7_E2alf. However, in populations previously vaccinated with live attenuated vaccines like the C-strain, passive immunity through maternal antibodies can interfere with efficacy of CP7_E2alf vaccination. Therefore, the efficacy of CP7_E2alf was examined in piglets from sows vaccinated once intramuscularly with C-strain vaccine 4 weeks before farrowing. Thus, these piglets were vaccinated intramuscularly with CP7_E2alf at the age of 5 or 8 weeks. Subsequently, the piglets and their mock-vaccinated littermate controls were challenged 2 weeks post vaccination with highly virulent Classical swine fever virus (CSFV) strain “Koslov”.     

Hypervariable antigenic region 1 of classical swine fever virus E2 protein impacts antibody neutralization

Envelope glycoprotein E2 of classical swine fever virus (CSFV) is the major antigen that induces neutralizing antibodies and confers protection against CSFV infection. There are three hypervariable antigenic regions (HAR1, HAR2 and HAR3) of E2 that are different between the group 1 vaccine C-strain and group 2 clinical isolates. This study was aimed to characterize the antigenic epitope region recognized by monoclonal antibody 4F4 (mAb-4F4) that is present in the group 2 field isolate HZ1-08, but not in the C-strain, and examine its impact on neutralization titers when antisera from different recombinant viruses were cross-examined. Indirect ELISA with C-strain E2-based chimeric proteins carrying the three HAR regions showed that the mAb-4F4 bound to HAR1 from HZ1-08 E2, but not to HAR2 or HAR3, indicating that the specific epitope is located in the HAR1 region. Of the 6 major residues differences between C-strain and field isolates, Glu713 in the HAR1 region of strain HZ1-08 is critical for mAb-4F4 binding either at the recombinant protein level or using intact recombinant viruses carrying single mutations. C-strain-based recombinant viruses carrying the most antigenic part of E2 or HAR1 from strain HZ1-08 remained non-pathogenic to pigs and induced good antibody responses. By cross-neutralization assay, we observed that the anti-C-strain serum lost most of its neutralization capacity to RecC-HZ-E2 and QZ-14 (subgroup 2.1d field isolate in 2014), and vice versa. More importantly, the RecC-HAR1 virus remained competent in neutralizing ReC-HZ-E2 and QZ-14 strains without compromising the neutralization capability to the recombinant C-strain. Thus, we propose that chimeric C-strain carrying the HAR1 region of field isolates is a good vaccine candidate for classical swine fever.     

Enhancement of the immunogenicity of an alphavirus replicon-based DNA vaccine against classical swine fever by electroporation and coinjection with a plasmid expressing porcine interleukin 2

Alphavirus replicon-based DNA vaccines have emerged as a promising approach to generation of antigen-specific immune responses. However, due to their low immunogenicity, there is a need for other approaches to enhance the vaccine potency. In this study, electroporation (EP) and a plasmid expressing porcine interleukin 2 (IL-2) were used to improve the immunogenicity of an alphavirus replicon-based DNA vaccine pSFV1CS-E2 against classical swine fever (CSF). Pigs were immunized with pSFV1CS-E2 alone or together with IL-2 by EP or by simple intramuscular injection. The results showed that EP combined with IL-2 resulted in marked enhancement of E2-specific antibody responses. Moreover, CSFV-specific lymphocyte proliferation, IFN-γ and IL-4 responses were increased significantly in the pSFV1CS-E2+IL-2/EP group. Pigs immunized with pSFV1CS-E2 plus IL-2 by EP were completely protected from lethal challenge, which is comparable to the sterilizing immunity and full protection offered by the live attenuated vaccine C-strain and in contrast with the incomplete protection conferred by pSFV1CS-E2 without or with IL-2 or EP alone, as demonstrated by the presence of pathological changes or/and viral loads. We conclude that EP in combination with IL-2 can significantly improve the immunogenicity of the plasmid DNA vaccine.